Wuschel (wus) gene homologs

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a WUS protein. The invention also relates to the construction of a chimeric gene encoding all or a portion of the WUS protein, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the WUS protein in a transformed host cell.

RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/US00/26648, filed Sep. 28, 2000, which claims the benefit under 35U.S.C. §119(e) of U.S. Provisional Application 60/157,216, filed Oct. 1,1999, the disclosures of which are herein incorporated in their entiretyby reference.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodingWuschel (WUS) proteins in plants and seeds.

BACKGROUND OF THE INVENTION

Organ formation in plants occurs via the activity of apical meristems.Plant meristems contain a pool of stem cells, which are able toself-maintain, give rise to a variety of cell types including cellsrequired for organ initiation. The initiation and maintenance of stemcells and their integration into organ-forming meristems are thus thebasis for continuous plant development.

The Wuschel protein, designated hereafter as WUS, plays a key role inthe initiation and maintenance of the apical meristem, which contains apool of pluripotent stem cells (Endrizzi et al., 1996, Plant Journal10:967 979; Laux et al., 1996, Development 122:87 96; and Mayer et al.,1998, Cell 95:805 815). Arabidopsis plants mutant for the WUS genecontain stem cells that are misspecified and that appear to undergodifferentiation. WUS encodes a novel homeodomain protein, whichpresumably functions as a transcriptional regulator (Mayer et al., 1998,Cell 95:805 815). The stem cell population of Arabidopsis shootmeristems is believed to be maintained by a regulatory loop between theCLAVATA (CLV) genes which promote organ initiation and the WUS genewhich is required for stem cell identity, with the CLV genes repressingWUS at the transcript level, and WUS expression being sufficient toinduce meristem cell identity and the expression of the stem cell markerCLV3 (Brand et al. (2000) Science 289:617-619; Schoof et al. (2000) Cell100:635-644). Constitutive expression of WUS in Arabidopsis has beenrecently shown to lead to adventitious shoot proliferation from leaves(in planta) (Laux, T., Talk Presented at the XVI International BotanicalCongress Meeting, Aug. 17, 1999, St. Louis, Mo.).

There is a great deal of interest in identifying the genes that encodeproteins involved in development in plants, generally toward theobjective of altering plant growth and architecture. WUS represents onesuch gene. However, the WUS gene can also be used for the novelapplication of stimulating in vitro growth of plant tissue and improvingtransformation. In this manner, this gene can expand the range oftissues types targeted for transformation. Specifically, the WUS genemay be used to improve meristem transformation frequencies and couldresult in genotype independent transformation of many important cropssuch as maize, soybean and sunflower. Furthermore, transformation intomeristems would stimulate the formation of new apical initials reducingthe chimeric nature of the transgenic events. Lastly, ectopic expressioninto non-meristematic cells would stimulate adventive meristemformation. This could lead to transformation of non-traditional tissuessuch as leaves, leaf bases, stem tissue, etc. Alternatively,transformation of a more traditional target such as callus or thescutellum of immature embryos could promote a “non-traditional” growthresponse, i.e. meristems in place of somatic embryos. In addition, WUSmay also be used as a genetic marker for meristems. Accordingly, theavailability of nucleic acid sequences encoding all or a portion of aWUS protein would facilitate studies to better understand programmeddevelopment in plants, provide genetic tools to enhance the efficiencyof gene transfer into meristem tissue and help provide alternativetransformation methods in several important crops.

SUMMARY OF THE INVENTION

The present invention concerns an isolated polynucleotide comprising anucleotide sequence selected from the group consisting of: (a) a firstnucleotide sequence encoding a polypeptide of at least 50 amino acidshaving at least 70% identity based on the Clustal method of alignmentwhen compared to a polypeptide selected from the group consisting of SEQID NOs:2, 4, and 12, (b) a second nucleotide sequence encoding apolypeptide of at least 100 amino acids having at least 70% identitybased on the Clustal method of alignment when compared to a polypeptideselected from the group consisting of SEQ ID NOs:14, 16, 18, and 20, (c)a third nucleotide sequence encoding a polypeptide of at least 180 aminoacids having at least 70% identity based on the Clustal method ofalignment when compared to a polypeptide of SEQ ID NO:24, (d) a fourthnucleotide sequence encoding a polypeptide of at least 230 amino acidshaving at least 70% identity based on the Clustal method of alignmentwhen compared to a polypeptide of SEQ ID NO:22, (e) a fifth nucleotidesequence encoding a polypeptide of at least 100 amino acids having atleast 80% identity based on the Clustal method of alignment whencompared to a polypeptide selected from the group consisting of SEQ IDNOs:6, 8, and 10, and (f) a sixth nucleotide sequence comprising thecomplement of (a), (b), (c), (d), or (e).

In a second embodiment, it is preferred that the isolated polynucleotideof the claimed invention comprises a first nucleotide sequence whichcomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 that codes forthe polypeptide selected from the group consisting of SEQ ID NOs:2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, and 24.

In a third embodiment, this invention concerns an isolatedpolynucleotide comprising a nucleotide sequence of at least one of 60(preferably at least one of 40, most preferably at least one of 30)contiguous nucleotides derived from a nucleotide sequence selected fromthe group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, and 23 and the complement of such nucleotide sequences.

In a fourth embodiment, this invention relates to a chimeric genecomprising an isolated polynucleotide of the present invention operablylinked to at least one suitable regulatory sequence.

In a fifth embodiment, the present invention concerns an isolated hostcell comprising a chimeric gene of the present invention or an isolatedpolynucleotide of the present invention. The host cell may beeukaryotic, such as a yeast or a plant cell, or prokaryotic, such as abacterial cell. The present invention also relates to a virus,preferably a baculovirus, comprising an isolated polynucleotide of thepresent invention or a chimeric gene of the present invention.

In a sixth embodiment, the invention also relates to a process forproducing an isolated host cell comprising a chimeric gene of thepresent invention or an isolated polynucleotide of the presentinvention, the process comprising either transforming or transfecting anisolated compatible host cell with a chimeric gene or isolatedpolynucleotide of the present invention.

In a seventh embodiment, the invention concerns an isolated WUSpolypeptide selected from the group consisting of: (a) a polypeptide ofat least 50 amino acids having at least 70% identity based on theClustal method of alignment when compared to a polypeptide selected fromthe group consisting of SEQ ID NOs:2, 4, and 12, (b) a polypeptide of atleast 100 amino acids having at least 70% identity based on the Clustalmethod of alignment when compared to a polypeptide selected from thegroup consisting of SEQ ID NOs:14, 16, 18, and 20, (c) a polypeptide ofat least 180 amino acids having at least 70% identity based on theClustal method of alignment when compared to a polypeptide of SEQ IDNO:24, (d) a polypeptide of at least 230 amino acids having at least 70%identity based on the Clustal method of alignment when compared to apolypeptide of SEQ ID NO:22, and (e) a polypeptide of at least 100 aminoacids having at least 80% identity based on the Clustal method ofalignment when compared to a polypeptide selected from the groupconsisting of SEQ ID NOs:6, 8, and 10.

In an eighth embodiment, the invention relates to a method of selectingan isolated polynucleotide that affects the level of expression of a WUSpolypeptide or enzyme activity in a host cell, preferably a plant cell,the method comprising the steps of: (a) constructing an isolatedpolynucleotide of the present invention or an isolated chimeric gene ofthe present invention; (b) introducing the isolated polynucleotide orthe isolated chimeric gene into a host cell; (c) measuring the level ofthe WUS polypeptide or enzyme activity in the host cell containing theisolated polynucleotide; and (d) comparing the level of the WUSpolypeptide or enzyme activity in the host cell containing the isolatedpolynucleotide with the level of the WUS polypeptide or enzyme activityin the host cell that does not contain the isolated polynucleotide.

In a ninth embodiment, the invention concerns a method of obtaining anucleic acid fragment encoding a substantial portion of a WUSpolypeptide, preferably a plant WUS polypeptide, comprising the stepsof: synthesizing an oligonucleotide primer comprising a nucleotidesequence of at least one of 60 (preferably at least one of 40, mostpreferably at least one of 30) contiguous nucleotides derived from anucleotide sequence selected from the group consisting of SEQ ID NOs:1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23, and the complement of suchnucleotide sequences; and amplifying a nucleic acid fragment (preferablya cDNA inserted in a cloning vector) using the oligonucleotide primer.The amplified nucleic acid fragment preferably will encode a substantialportion of a WUS amino acid sequence.

In a tenth embodiment, this invention relates to a method of obtaining anucleic acid fragment encoding all or a substantial portion of the aminoacid sequence encoding a WUS polypeptide comprising the steps of:probing a cDNA or genomic library with an isolated polynucleotide of thepresent invention; identifying a DNA clone that hybridizes with anisolated polynucleotide of the present invention; isolating theidentified DNA clone; and sequencing the cDNA or genomic fragment thatcomprises the isolated DNA clone.

In an eleventh embodiment, this invention concerns a composition, suchas a hybridization mixture, comprising an isolated polynucleotide of thepresent invention.

In a twelfth embodiment, this invention concerns a method for positiveselection of a transformed cell comprising: (a) transforming a host cellwith the chimeric gene of the present invention or an expressioncassette of the present invention; and (b) growing the transformed hostcell, preferably a plant cell, such as a monocot or a dicot, underconditions which allow expression of the WUS polynucleotide in an amountsufficient to complement a null mutant to provide a positive selectionmeans.

In a thirteenth embodiment, this invention relates to a method ofaltering the level of expression of a WUS protein in a host cellcomprising: (a) transforming a host cell with a chimeric gene of thepresent invention; and (b) growing the transformed host cell underconditions that are suitable for expression of the chimeric gene whereinexpression of the chimeric gene results in production of altered levelsof the WUS protein in the transformed host cell.

BRIEF DESCRIPTION OF THE DRAWING AND SEQUENCE LISTINGS

The invention can be more fully understood from the following detaileddescription and the accompanying drawing and Sequence Listing which forma part of this application.

FIG. 1 shows an alignment of the amino acid sequences of WUS proteinencoded by the nucleotide sequences derived from corn clonecpilc.pk012.p19 (SEQ ID NO:4), corn clone p0058.chpab57r (SEQ ID NO:10),soybean clone ses4d.pk0033.c8 (SEQ ID NO:20), soybean clonesgs5c.pk0002.f2 (SEQ ID NO:22), and a contig assembled using soybeanclone ssm.pk0060.h4 and NCBI GenBank Identifier (GI) No. 4395781 (SEQ IDNO:24), and the WUS protein from Arabidopsis thaliana (NCBI GI No.4090200; SEQ ID NO:25). Amino acids which are conserved among all and atleast two sequences with an amino acid at that position are indicatedwith an asterisk (*). Dashes are used by the program to maximizealignment of the sequences.

Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. Table 1 also identifies the cDNA clonesas individual ESTs (“EST”), the sequences of the entire cDNA insertscomprising the indicated cDNA clones (“FIS”), contigs assembled from twoor more ESTs (“Contig”), contigs assembled from an FIS and one or moreESTs or PCR fragment sequence (“Contig*”), or sequences encoding theentire protein derived from an EST, an FIS, a contig, or an FIS and PCRfragment sequence (“CGS”). Nucleotide SEQ ID NOs:1, 5, 11, and 15correspond to nucleotide SEQ ID NOs:1, 3, 5, and 7, respectively,presented in U.S. Provisional Application No. 60/157,216, filed Oct. 1,1999. Amino acid SEQ ID NOs:2, 6, 12, and 16 correspond to amino acidSEQ ID NOs:2, 4, 6, and 8, respectively, presented in U.S. ProvisionalApplication No. 60/157,216, filed Oct. 1, 1999. The sequencedescriptions and Sequence Listing attached hereto comply with the rulesgoverning nucleotide and/or amino acid sequence disclosures in patentapplications as set forth in 37C.F.R. §1.821-1.825.

TABLE 1 WUS Proteins SEQ ID NO: Protein (Nucleo- (Amino (Plant Source)Clone Designation Status tide) Acid) WUS Protein Contig of Contig 1 2(Corn) cpg1c.pk006.b16 cpi1c.pk012.p19 WUS Protein cpi1c.pk012.p19 (FIS)CGS 3 4 (Corn) WUS Protein p0016.ctsas50r EST 5 6 (Corn) WUS Proteinp0016.ctsas50r FIS 7 8 (Corn) WUS Protein p0058.chpab57r (FIS) CGS 9 10(Corn) WUS Protein p0083.cldev71r EST 11 12 (Corn) WUS Proteinp0083.cldev71r FIS 13 14 (Corn) WUS Protein Contig of Contig 15 16(Soybean) scr1c.pk001.d2 ses4d.pk0033.c8 WUS Protein scr1c.pk001.d2 FIS17 18 (Soybean) WUS Protein ses4d.pk0033.c8 (FIS) CGS 19 20 (Soybean)WUS Protein sgs5c.pk0002.f2 (EST) CGS 21 22 (Soybean) WUS Protein Contigof CGS 23 24 (Soybean) ssm.pk0060.h4 (FIS) NCBI GI No. 4395781

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidsequence”, and “nucleic acid fragment”/“isolated nucleic acid fragment”are used interchangeably herein. These terms encompass nucleotidesequences and the like. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof. An isolatedpolynucleotide of the present invention may include at least one of 60contiguous nucleotides, preferably at least one of 40 contiguousnucleotides, most preferably one of at least 30 contiguous nucleotidesderived from SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23, orthe complement of such sequences.

The term “isolated” polynucleotide refers to a polynucleotide that issubstantially free from other nucleic acid sequences, such as otherchromosomal and extrachromosomal DNA and RNA, that normally accompany orinteract with it as found in its naturally occurring environment.Isolated polynucleotides may be purified from a host cell in which theynaturally occur. Conventional nucleic acid purification methods known toskilled artisans may be used to obtain isolated polynucleotides. Theterm also embraces recombinant polynucleotides and chemicallysynthesized polynucleotides.

The term “recombinant” means, for example, that a nucleic acid sequenceis made by an artificial combination of two otherwise separated segmentsof sequence, e.g., by chemical synthesis or by the manipulation ofisolated nucleic acids by genetic engineering techniques.

As used herein, “contig” refers to a nucleotide sequence that isassembled from two or more constituent nucleotide sequences that sharecommon or overlapping regions of sequence homology. For example, thenucleotide sequences of two or more nucleic acid fragments can becompared and aligned in order to identify common or overlappingsequences. Where common or overlapping sequences exist between two ormore nucleic acid fragments, the sequences (and thus their correspondingnucleic acid fragments) can be assembled into a single contiguousnucleotide sequence.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough for example antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-à-vis the ability to mediategene silencing or alteration of the functional properties of theresulting protein molecule. It is therefore understood that theinvention encompasses more than the specific exemplary nucleotide oramino acid sequences and includes functional equivalents thereof. Theterms “substantially similar” and “corresponding substantially” are usedinterchangeably herein.

Substantially similar nucleic acid fragments may be selected byscreening nucleic acid fragments representing subfragments ormodifications of the nucleic acid fragments of the instant invention,wherein one or more nucleotides are substituted, deleted and/orinserted, for their ability to affect the level of the polypeptideencoded by the unmodified nucleic acid fragment in a plant or plantcell. For example, a substantially similar nucleic acid fragmentrepresenting at least one of 30 contiguous nucleotides derived from theinstant nucleic acid fragment can be constructed and introduced into aplant or plant cell. The level of the polypeptide encoded by theunmodified nucleic acid fragment present in a plant or plant cellexposed to the substantially similar nucleic fragment can then becompared to the level of the polypeptide in a plant or plant cell thatis not exposed to the substantially similar nucleic acid fragment.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby using nucleic acid fragments that do not share 100% sequence identitywith the gene to be suppressed. Moreover, alterations in a nucleic acidfragment which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts. Consequently, an isolated polynucleotide comprising anucleotide sequence of at least one of 60 (preferably at least one of40, most preferably at least one of 30) contiguous nucleotides derivedfrom a nucleotide sequence selected from the group consisting of SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23, and the complement ofsuch nucleotide sequences may be used in methods of selecting anisolated polynucleotide that affects the expression of a WUS polypeptidein a host cell. A method of selecting an isolated polynucleotide thataffects the level of expression of a polypeptide in a virus or in a hostcell (eukaryotic, such as plant or yeast, prokaryotic such as bacterial)may comprise the steps of: constructing an isolated polynucleotide ofthe present invention or an isolated chimeric gene of the presentinvention; introducing the isolated polynucleotide or the isolatedchimeric gene into a host cell; measuring the level of a polypeptide orenzyme activity in the host cell containing the isolated polynucleotide;and comparing the level of a polypeptide or enzyme activity in the hostcell containing the isolated polynucleotide with the level of apolypeptide or enzyme activity in a host cell that does not contain theisolated polynucleotide.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. One set of preferred conditionsuses a series of washes starting with 6×SSC, 0.5% SDS at roomtemperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30min. A more preferred set of stringent conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of highly stringent conditionsuses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Suitable nucleic acid fragments (isolated polynucleotides of thepresent invention) encode polypeptides that are at least about 70%identical, preferably at least about 80% identical to the amino acidsequences reported herein. Preferred nucleic acid fragments encode aminoacid sequences that are about 85% identical to the amino acid sequencesreported herein. More preferred nucleic acid fragments encode amino acidsequences that are at least about 90% identical to the amino acidsequences reported herein. Most preferred are nucleic acid fragmentsthat encode amino acid sequences that are at least about 95% identicalto the amino acid sequences reported herein. Suitable nucleic acidfragments not only have the above identities but typically encode apolypeptide having at least 50 amino acids, preferably at least 100amino acids, more preferably at least 150 or 180 amino acids, still morepreferably at least 200 or 230 amino acids, and most preferably at least250 amino acids. Sequence alignments and percent identity calculationswere performed using the Megalign program of the LASERGENEbioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiplealignment of the sequences was performed using the Clustal method ofalignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parametersfor pairwise alignments using the Clustal method were KTUPLE 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to a nucleic acidfragment, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of nucleic acid fragments may be accomplishedusing well established procedures, or automated chemical synthesis canbe performed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of the nucleotide sequence to reflectthe codon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign-gene” refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or may be composed ofdifferent elements derived from different promoters found in nature, ormay even comprise synthetic nucleotide segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene in different tissues or cell types, or at differentstages of development, or in response to different environmentalconditions. Promoters which cause a nucleic acid fragment to beexpressed in most cell types at most times are commonly referred to as“constitutive promoters”. New promoters of various types useful in plantcells are constantly being discovered; numerous examples may be found inthe compilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

“Translation leader sequence” refers to a nucleotide sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

“3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptides by the cell. “cDNA” refers to DNA that is complementary toand derived from an mRNA template. The cDNA can be single-stranded orconverted to double stranded form using, for example, the Klenowfragment of DNA polymerase I. “Sense-RNA” refers to an RNA transcriptthat includes the mRNA and so can be translated into a polypeptide bythe cell. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single polynucleotide so that the functionof one is affected by the other. For example, a promoter is operablylinked with a coding sequence when it is capable of affecting theexpression of that coding sequence (i.e., that the coding sequence isunder the transcriptional control of the promoter). Coding sequences canbe operably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

A “protein” or “polypeptide” is a chain of amino acids arranged in aspecific order determined by the coding sequence in a polynucleotideencoding the polypeptide. Each protein or polypeptide has a uniquefunction.

“Altered levels” or “altered expression” refers to the production ofgene product(s) in transgenic organisms in amounts or proportions thatdiffer from that of normal or non-transformed organisms.

“Null mutant” refers here to a host cell which either lacks theexpression of a certain polypeptide or expresses a polypeptide which isinactive or does not have any detectable expected enzymatic function.

“Mature protein” or the term “mature” when used in describing a proteinrefers to a post-translationally processed polypeptide; i.e., one fromwhich any pre- or propeptides present in the primary translation producthave been removed. “Precursor protein” or the term “precursor” when usedin describing a protein refers to the primary product of translation ofmRNA; i.e., with pre- and propeptides still present. Pre- andpropeptides may be but are not limited to intracellular localizationsignals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Thus, isolated polynucleotides of thepresent invention can be incorporated into recombinant constructs,typically DNA constructs, capable of introduction into and replicationin a host cell. Such a construct can be a vector that includes areplication system and sequences that are capable of transcription andtranslation of a polypeptide-encoding sequence in a given host cell. Anumber of vectors suitable for stable transfection of plant cells or forthe establishment of transgenic plants have been described in, e.g.,Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987;Weissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989; and Flevin et al., Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990. Typically, plant expression vectors include,for example, one or more cloned plant genes under the transcriptionalcontrol of 5′ and 3′ regulatory sequences and a dominant selectablemarker. Such plant expression vectors also can contain a promoterregulatory region (e.g., a regulatory region controlling inducible orconstitutive, environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

“PCR” or “polymerase chain reaction” is well known by those skilled inthe art as a technique used for the amplification of specific DNAsegments (U.S. Pat. Nos. 4,683,195 and 4,800,159).

The present invention concerns an isolated polynucleotide comprising anucleotide sequence selected from the group consisting of: (a) a firstnucleotide sequence encoding a polypeptide of at least 50 amino acidshaving at least 70% identity based on the Clustal method of alignmentwhen compared to a polypeptide selected from the group consisting of SEQID NOs:2, 4, and 12, (b) a second nucleotide sequence encoding apolypeptide of at least 100 amino acids having at least 70% identitybased on the Clustal method of alignment when compared to a polypeptideselected from the group consisting of SEQ ID NOs:14, 16, 18, and 20, (c)a third nucleotide sequence encoding a polypeptide of at least 180 aminoacids having at least 70% identity based on the Clustal method ofalignment when compared to a polypeptide of SEQ ID NO:24, (d) a fourthnucleotide sequence encoding a polypeptide of at least 230 amino acidshaving at least 70% identity based on the Clustal method of alignmentwhen compared to a polypeptide of SEQ ID NO:22, (e) a fifth nucleotidesequence encoding a polypeptide of at least 100 amino acids having atleast 80% identity based on the Clustal method of alignment whencompared to a polypeptide selected from the group consisting of SEQ IDNOs:6, 8, and 10, and (f) a sixth nucleotide sequence comprising thecomplement of (a), (b), (c), (d), or (e).

Preferably, the first nucleotide sequence comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, and 23, that codes for the polypeptide selectedfrom the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, and 24.

Nucleic acid fragments encoding at least a portion of several WUSproteins have been isolated and identified by comparison of random plantcDNA sequences to public databases containing nucleotide and proteinsequences using the BLAST algorithms well known to those skilled in theart. The nucleic acid fragments of the instant invention may be used toisolate cDNAs and genes encoding homologous proteins from the same orother plant species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g., polymerase chain reaction, ligase chain reaction).

For example, genes encoding other WUS proteins, either as cDNAs orgenomic DNAs, could be isolated directly by using all or a portion ofthe instant nucleic acid fragments as DNA hybridization probes to screenlibraries from any desired plant employing methodology well known tothose skilled in the art. Specific oligonucleotide probes based upon theinstant nucleic acid sequences can be designed and synthesized bymethods known in the art (Maniatis). Moreover, an entire sequence can beused directly to synthesize DNA probes by methods known to the skilledartisan such as random primer DNA labeling, nick translation,end-labeling techniques, or RNA probes using available in vitrotranscription systems. In addition, specific primers can be designed andused to amplify a part or all of the instant sequences. The resultingamplification products can be labeled directly during amplificationreactions or labeled after amplification reactions, and used as probesto isolate full length cDNA or genomic fragments under conditions ofappropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the mRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002)to generate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220).Products generated by the 3′ and 5′ RACE procedures can be combined togenerate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165).Consequently, a polynucleotide comprising a nucleotide sequence of atleast one of 60 (preferably one of at least 40, most preferably one ofat least 30) contiguous nucleotides derived from a nucleotide sequenceselected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, and 23 and the complement of such nucleotide sequencesmay be used in such methods to obtain a nucleic acid fragment encoding asubstantial portion of an amino acid sequence of a polypeptide.

The present invention relates to a method of obtaining a nucleic acidfragment encoding a substantial portion of a WUS polypeptide, preferablya substantial portion of a plant WUS polypeptide, comprising the stepsof: synthesizing an oligonucleotide primer comprising a nucleotidesequence of at least one of 60 (preferably at least one of 40, mostpreferably at least one of 30) contiguous nucleotides derived from anucleotide sequence selected from the group consisting of SEQ ID NOs:1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23, and the complement of suchnucleotide sequences; and amplifying a nucleic acid fragment (preferablya cDNA inserted in a cloning vector) using the oligonucleotide primer.The amplified nucleic acid fragment preferably will encode a portion ofa WUS polypeptide.

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol.36:1-34; Maniatis).

In another embodiment, this invention concerns viruses and host cellscomprising either the chimeric genes of the invention as describedherein or an isolated polynucleotide of the invention as describedherein. Examples of host cells which can be used to practice theinvention include, but are not limited to, yeast, bacteria, and plants.

As was noted above, the nucleic acid fragments of the instant inventionmay be used to create transgenic plants in which the disclosedpolypeptides are present at higher or lower levels than normal or incell types or developmental stages in which they are not normally found.This would have the effect of altering development (e.g., the initiationand maintenance of meristem apical initials) in those plants.

Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.The chimeric gene may comprise promoter sequences and translation leadersequences derived from the same genes. 3′ non-coding sequences encodingtranscription termination signals may also be provided. The instantchimeric gene may also comprise one or more introns in order tofacilitate gene expression.

Plasmid vectors comprising the instant isolated polynucleotide (orchimeric gene) may be constructed. The choice of plasmid vector isdependent upon the method that will be used to transform host plants.The skilled artisan is well aware of the genetic elements that must bepresent on the plasmid vector in order to successfully transform, selectand propagate host cells containing the chimeric gene. The skilledartisan will also recognize that different independent transformationevents will result in different levels and patterns of expression (Joneset al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen.Genetics 218:78-86), and thus that multiple events must be screened inorder to obtain lines displaying the desired expression level andpattern. Such screening may be accomplished by Southern analysis of DNA,Northern analysis of mRNA expression, Western analysis of proteinexpression, or phenotypic analysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by directing the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys. 100: 1627-1632) with or without removingtargeting sequences that are already present. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of use may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression of aspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppression technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent chimeric genes utilizing different regulatory elements knownto the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individualtransgenics for those that most effectively display the desiredphenotype. Accordingly, the skilled artisan will develop methods forscreening large numbers of transformants. The nature of these screenswill generally be chosen on practical grounds. For example, one canscreen by looking for changes in gene expression by using antibodiesspecific for the protein encoded by the gene being suppressed, or onecould establish assays that specifically measure enzyme activity. Apreferred method will be one which allows large numbers of samples to beprocessed rapidly, since it will be expected that a large number oftransformants will be negative for the desired phenotype.

In another embodiment, the present invention concerns an isolatedpolypeptide selected from the group consisting of: (a) a polypeptide ofat least 50 amino acids having at least 70% identity based on theClustal method of alignment when compared to a polypeptide selected fromthe group consisting of SEQ ID NOs:2, 4, and 12, (b) a polypeptide of atleast 100 amino acids having at least 70% identity based on the Clustalmethod of alignment when compared to a polypeptide selected from thegroup consisting of SEQ ID NOs:14, 16, 18, and 20, (c) a polypeptide ofat least 180 amino acids having at least 70% identity based on theClustal method of alignment when compared to a polypeptide of SEQ IDNO:24, (d) a polypeptide of at least 230 amino acids having at least 70%identity based on the Clustal method of alignment when compared to apolypeptide of SEQ ID NO:22, and (e) a polypeptide of at least 100 aminoacids having at least 80% identity based on the Clustal method ofalignment when compared to a polypeptide selected from the groupconsisting of SEQ ID NOs:6, 8, and 10.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to these proteins by methods wellknown to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded WUS protein. An example of a vector for high levelexpression of the instant polypeptides in a bacterial host is provided(Example 12).

All or a substantial portion of the polynucleotides of the instantinvention may also be used as probes for genetically and physicallymapping the genes that they are a part of, and used as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4:3741. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: APractical Guide, Academic press 1996, pp. 319-346, and references citedtherein).

In another embodiment, nucleic acid probes derived from the instantnucleic acid sequences may be used in direct fluorescence in situhybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).Although current methods of FISH mapping favor use of large clones(several to several hundred KB; see Laan et al. (1995) Genome Res.5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med. 11:95 96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325 332),allele-specific ligation (Landegren et al. (1988) Science 241:10771080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res.18:3671), Radiation Hybrid Mapping (Walter et al. (1994) Nat. Genet.7:22 28) and Happy Mapping (Dear and Cook (1989) Nucleic Acids Res.17:6795 6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell7:75-84). The latter approach may be accomplished in two ways. First,short segments of the instant nucleic acid fragments may be used inpolymerase chain reaction protocols in conjunction with a mutation tagsequence primer on DNAs prepared from a population of plants in whichMutator transposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptide.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptide can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

EXAMPLES

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. Thus, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

The disclosure of each reference set forth herein is incorporated hereinby reference in its entirety.

Example 1 Composition of cDNA Libraries Isolation and Sequencing of cDNAClones

cDNA libraries representing mRNAs from various corn (Zea mays) andsoybean (Glycine max) tissues were prepared. The characteristics of thelibraries are described below.

TABLE 2 cDNA Libraries from Corn and Soybean Library Tissue Clone cpg1cCorn Pooled BMS Treated with Chemicals cpg1c.pk006.b16 Related to RNA,DNA Synthesis* cpi1c Corn Pooled BMS Treated with Chemicalscpi1c.pk012.p19 Related to Biochemical Compound Synthesis** p0016 CornTassel Shoots, Pooled, 0.1-1.4 cm p0016.ctsas50r p0058 Sweet Corn Hybrid(Honey N Pearl) p0058.chpab57r Shoot Culture p0083 Corn Whole Kernels 7Days After p0083.cldev71r Pollination scr1c Soybean EmbryogenicSuspension Culture scr1c.pk001.d2 Subjected to 4 Vacuum Cycles andCollected 12 Hrs Later ses4d Soybean Embryogenic Suspension 4 Daysses4d.pk0033.c8 After Subculture sgs5c Soybean Seeds 4 Days AfterGermination sgs5c.pk0002.f2 ssm Soybean Shoot Meristem ssm.pk0060.h4*Chemicals used included hydroxyurea, aphidicolin, HC-toxin, actinomysinD, all of which are commercially available from Calbiochem-NovabiochemCorp. (1-800-628-8470) **Chemicals used included sorbitol, egosterol,taxifolin, methotrexate, D-mannose, D-glactose, alpha-amino adipic acid,ancymidol, all of which are commercially available fromCalbiochem-Novabiochem Corp. (1-800-628-8470)

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the cDNA libraries in Uni-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The Uni-ZAP™ XR libraries are converted into plasmid libraries accordingto the protocol provided by Stratagene. Upon conversion, cDNA insertswill be contained in the plasmid vector pBluescript. In addition, thecDNAs may be introduced directly into precut Bluescript II SK(+) vectors(Stratagene) using T4 DNA ligase (New England Biolabs), followed bytransfection into DH10B cells according to the manufacturer's protocol(GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors,plasmid DNAs are prepared from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids, or the insert cDNAsequences are amplified via polymerase chain reaction using primersspecific for vector sequences flanking the inserted cDNA sequences.Amplified insert DNAs or plasmid DNAs are sequenced in dye-primersequencing reactions to generate partial cDNA sequences (expressedsequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Full-insert sequence (FIS) data is generated utilizing a modifiedtransposition protocol. Clones identified for FIS are recovered fromarchived glycerol stocks as single colonies, and plasmid DNAs areisolated via alkaline lysis. Isolated DNA templates are reacted withvector primed M13 forward and reverse oligonucleotides in a PCR-basedsequencing reaction and loaded onto automated sequencers. Confirmationof clone identification is performed by sequence alignment to theoriginal EST sequence from which the FIS request is made.

Confirmed templates are transposed via the Primer Island transpositionkit (PE Applied Biosystems, Foster City, Calif.) which is based upon theSaccharomyces cerevisiae Ty1 transposable element (Devine and Boeke(1994) Nucleic Acids Res. 22:3765-3772). The in vitro transpositionsystem places unique binding sites randomly throughout a population oflarge DNA molecules. The transposed DNA is then used to transform DH10Belectro-competent cells (Gibco BRL/Life Technologies, Rockville, Md.)via electroporation. The transposable element contains an additionalselectable marker (named DHFR; Fling and Richards (1983) Nucleic AcidsRes. 11:5147-5158), allowing for dual selection on agar plates of onlythose subclones containing the integrated transposon. Multiple subclonesare randomly selected from each transposition reaction, plasmid DNAs areprepared via alkaline lysis, and templates are sequenced (ABI Prismdye-terminator ReadyReaction mix) outward from the transposition eventsite, utilizing unique primers specific to the binding sites within thetransposon.

Sequence data is collected (ABI Prism Collections) and assembled usingPhred/Phrap (P. Green, University of Washington, Seattle). Phrep/Phrapis a public domain software program which re-reads the ABI sequencedata, re-calls the bases, assigns quality values, and writes the basecalls and quality values into editable output files. The Phrap sequenceassembly program uses these quality values to increase the accuracy ofthe assembled sequence contigs. Assemblies are viewed by the Consedsequence editor (D. Gordon, University of Washington, Seattle).

Example 2 Identification of cDNA Clones

cDNA clones encoding WUS protein were identified by conducting BLAST(Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol.215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches forsimilarity to sequences contained in the BLAST “nr” database (comprisingall non-redundant GenBank CDS translations, sequences derived from the3-dimensional structure Brookhaven Protein Data Bank, the last majorrelease of the SWISS-PROT protein sequence database, EMBL, and DDBJdatabases). The cDNA sequences obtained in Example 1 were analyzed forsimilarity to all publicly available DNA sequences contained in the “nr”database using the BLASTN algorithm provided by the National Center forBiotechnology Information (NCBI). The DNA sequences were translated inall reading frames and compared for similarity to all publicly availableprotein sequences contained in the “nr” database using the BLASTXalgorithm (Gish and States (1993) Nat. Genet. 3:266-272) provided by theNCBI. For convenience, the P-value (probability) of observing a match ofa cDNA sequence to a sequence contained in the searched databases merelyby chance as calculated by BLAST are reported herein as “pLog” values,which represent the negative of the logarithm of the reported P-value.Accordingly, the greater the pLog value, the greater the likelihood thatthe cDNA sequence and the BLAST “hit” represent homologous proteins.

ESTs submitted for analysis are compared to the genbank database asdescribed above. ESTs that contain sequences more 5- or 3-prime can befound by using the BLASTn algorithm (Altschul et al (1997) Nucleic AcidsRes. 25:3389-3402.) against the Du Pont proprietary database comparingnucleotide sequences that share common or overlapping regions ofsequence homology. Where common or overlapping sequences exist betweentwo or more nucleic acid fragments, the sequences can be assembled intoa single contiguous nucleotide sequence, thus extending the originalfragment in either the 5 or 3 prime direction. Once the most 5-prime ESTis identified, its complete sequence can be determined by Full InsertSequencing as described in Example 1. Homologous genes belonging todifferent species can be found by comparing the amino acid sequence of aknown gene (from either a proprietary source or a public database)against an EST database using the tBLASTn algorithm. The tBLASTnalgorithm searches an amino acid query against a nucleotide databasethat is translated in all 6 reading frames. This search allows fordifferences in nucleotide codon usage between different species, and forcodon degeneracy.

Example 3 Characterization of cDNA Clones Encoding WUS Protein Homologs

The BLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs to WUSproteins from Arabidopsis thaliana (NCBI GenBank Identifier (GI) No.3785979) and Arabidopsis thaliana (NCBI GI No. 4090200). Shown in Table3 are the BLAST results for individual ESTs (“EST”), the sequences ofthe entire cDNA inserts comprising the indicated cDNA clones (“FIS”), orcontigs assembled from two or more ESTs (“Contig”):

TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous toArabidopsis thaliana WUS Proteins Clone Status BLAST pLog Score Contigcomposed of: Contig 14.30 (NCBI GI No. 3785979) cpg1c.pk006.b16cpi1c.pk012.p19 p0016.ctsas50r EST 31.00 (NCBI GI No. 4090200)p0083.cldev71r EST 17.40 (NCBI GI No. 3785979) Contig composed of:Contig 24.52 (NCBI GI No. 3785979) scr1c.pk001.d2 ses4d.pk0033.c8

The data in Table 4 represents a calculation of the percent identity ofthe amino acid sequences set forth in SEQ ID NOs:2, 6, 12 and 16 and theArabidopsis thaliana (NCBI GI No. 3785979) and (NCBI GI No. 4090200)sequences (SEQ ID NOs:27 and 28 respectively). The percent identitybetween the amino acid sequences set forth in SEQ ID NOs: 2, 6, 12 and16 ranged from 35-40%.

TABLE 4 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toArabidopsis thaliana WUS Proteins SEQ ID NO. Percent Identity to 2 43%(NCBI GI No. 3785979) 6 45% (NCBI GI No. 4090200) 12 37% (NCBI GI No.3785979) 16 37% (NCBI GI No. 3785979)

The sequence of the entire cDNA insert in most of the clones listed inTable 3 was determined. Further sequencing and searching of the DuPontproprietary database allowed the identification of other corn andsoybean clones encoding WUS protein. The BLASTX search using the ESTsequences from clones listed in Table 5 revealed similarity of thepolypeptides encoded by the cDNAs to WUS proteins from Arabidopsisthaliana (NCBI GI Nos. 3785979, 4090200, 4580396, 9294502 and 6091768)and Oryza sativa (NCBI GI No. 8099120). Shown in Table 5 are the BLASTresults for individual ESTs (“EST”), the sequences of the entire cDNAinserts comprising the indicated cDNA clones (“FIS”), sequences ofcontigs assembled from two or more ESTs (“Contig”), sequences of contigsassembled from an FIS and one or more ESTs or PCR fragment sequence(“Contig*”), or sequences encoding the entire protein derived from anEST, an FIS, a contig, or an FIS and PCR fragment sequence (“CGS”):

TABLE 5 BLAST Results for Sequences Encoding Polypeptides Homologous toWUS Proteins BLAST Results Clone Status NCBI GI No. BLAST pLog Scorecpi1c.pk012.p19 (FIS) CGS 3785979 21.30 p0016.ctsas50r FIS 4090200 27.00p0058.chpab57r (FIS) CGS 6091768 36.52 p0083.cldev71r FIS 4580396 15.70scr1c.pk001.d2 FIS 3785979 20.04 ses4d.pk0033.c8 (FIS) CGS 3785979 21.10sgs5c.pk0002.f2 (EST) CGS 8099120 23.70 Contig of CGS 9294502 23.00ssm.pk0060.h4 (FIS) NCBI GI No. 4395781

FIG. 1 presents an alignment of the amino acid sequences set forth inSEQ ID NOs:4, 10, 20, 22, and 24 and the Arabidopsis thaliana sequence(NCBI GI No. 4090200; SEQ ID NO:25). The data in Table 6 represents acalculation of the percent identity of the amino acid sequences setforth in SEQ ID NOs:4, 10, 20, 22, and 24 and the Arabidopsis thalianasequence (NCBI GI No. 4090200; SEQ ID NO:25).

TABLE 6 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toWUS Protein Percent Identity to SEQ ID NO. NCBI GI No. 4090200; SEQ IDNO:25 4 22.7 10 18.2 20 25.0 22 21.6 24 22.2

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of a WUS protein. These sequences representthe first corn and soybean sequences encoding WUS proteins known toApplicant.

Example 4 Sunflower Meristem Transformation

There are a number of published examples of meristem transformationsystems for dicot species including soybean (McCabe et al, 1988),sunflower (Bidney et al., 1992), and cotton (Gould et al., 1998), wherechimeric genes are delivered to cells of the meristem and thenparticipate in formation of shoots, reproductive structures andultimately seed. Transgene delivery is accomplished by both standardparticle bombardment protocols as described for soybean or by T-DNA andAgrobacterium protocols as described for sunflower and cotton. The WUSgene could be delivered to dicot meristem targets for either stable ortransient transformation to impact the transformation response. WUScould be delivered together with agronomic genes or be used as aconditioning treatment prior to or following the protocol for DNAdelivery. The methods for sunflower meristem transformation follow.

Sunflower meristem transformation is achieved by a protocol for directDNA delivery by particle bombardment or a protocol involving acombination of DNA-free particle bombardment followed by use ofAgrobacterium inoculation for DNA delivery as described in Bidney et al.(supra). Sunflower line SMF3, described in Burrus et al. (1991, PlantCell Rep. 10:161 166) is used. The explant source is dry sunflower seedthat is imbibed and dissected into meristem explants. Seeds are dehulledand surface sterilized then placed in sterile petri plates on two layersof filter paper moistened with sterile distilled water for overnightimbibition in the dark at 26° C. in a Percival incubator. The next day,cotyledons and root radicle are removed and meristem explantstransferred to 374E medium (MS salts, Shepard vitamins, 40 mg/l adeninesulfate, 3% sucrose, 0.5 mg/l 6 BAP, 0.25 mg/l IAA, 0.1 mg/l GA, pH 5.6,and 0.8% Phytagar). Explants are cultured for 24 hr on 374E medium inthe dark at 26° C. Following this culture period, elongated primaryleaves are removed to expose the apical meristem. The meristem explantsare placed in the center of petri plates with 374M medium (374E with1.2% Phytagar) in preparation for particle bombardment then back in thedark for another 24 hr period at 26° C.

Particle preparation for the Agrobacterium based protocol is done bysuspending 18.8 mg of 1.8 um tungsten particles or 21.6 mg of 2.0 μmgold particles in 200 μl absolute ethanol. Following particleresuspension by sonication and vigorous mixing, 10 μl of particlesuspension is dropped on the center of the surface of macro-carrier.Plates of 374M medium containing sunflower meristem explants are shottwice by a DuPont Biolistics PDS1000 helium gun with vacuum drawn to 26Hg, with 650 psi rupture discs, and at the top level in the gun.Following particle bombardment, explants are spread out on the 374Mplates, inoculated with an Agrobacterium suspension and co-cultured inthe light at 26° C. for 4d. The Agrobacterium inoculating suspension isprepared by first starting a 5 ml liquid culture in 60A medium withkanamycin (YEP medium—10 g/l Bactopeptone, 10 g/l yeast extract, 5 g/lsodium chloride, pH 7.0, and 50 mg/l kanamycin) grown to log phase(OD600 0.5-1.0). The log phase growth Agrobacterium suspension iscentrifuged at 6K for 5 min and the supernatant discarded. The bacterialpellet is resuspended in inoculation medium (IM) (IM—12.5 mM MES, 1 g/lammonium chloride, 0.3 g/1 magnesium sulfate, pH 5.7) to a finalcalculated OD600 vis of 4.0. The inoculating Agrobacterium suspension isapplied twice using a micro-pipette and 0.5 ul of suspension perexplant. After the 4 d co-cultivation of sunflower meristem explants,the expanded bases of explants are trimmed off and they are transferredto 374C medium (374E which lacks hormones, but adds 250 mg/l cefotaxime)and cultured for two weeks in the light under 18 hr day length at 26° C.

Alternatively, a direct DNA delivery protocol can be applied tosunflower meristem explants prepared as described above. Particles areprepared as follows: to 50 μL of a 15 mg/mL 0.6 μm gold particlesuspension is added (in order): 10 μL DNA (0.1 μg/uL), 20 μL spermidine(0.1 M), and 50 μL CaCl₂ (2.5 M). The particle preparation is thenagitated for three minutes, spun in a microfuge for 10 seconds and thesupernatant removed. The DNA-coated particles are then washed once in500 μL 100% ethanol and resuspended in 30 uL of 100% ethanol. TheDNA/particle suspension can be sonicated three times for one secondeach. Five μL of the DNA-coated gold particles are then loaded on eachmacro carrier disk. Meristem explants are bombarded as described in theprevious paragraph, spread out on 374M medium, and cultured for 4 d in aPercival incubator under 18 hr of daylength at 26° C. The expanded basesof the explant are then cut off and the explant transferred to 374Cmedium for 2 wk of culture under the long day conditions at 26° C.

After two weeks sunflower shoots emerge from the meristem explants on374C medium. The shoots can be scored destructively or non-destructivelyfor the frequency of transgenic sectors per experiment and the qualityof sectors with longer, wider, and deeper transgenic sectors being moredesirable. They can be scored and compared to control using scorablemarkers such as the GUS enzyme or green fluorescence protein (GFP).Transgenic plants and seed can be obtained by adding steps to theprocedure as outlined below. An assay is required such as an enzymeassay or ELISA for an agronomic protein of interest. An example isprovided using the enzyme oxalate oxidase as a scorable marker. Chemicalselection is not required for this transformation process.

Primary shoots following two weeks of culture on 374C medium arescreened using the oxalate oxidase enzyme assay. Oxalate oxidase enzymeassays were set up using fresh leaf or cotyledon tissue to identifytransformants. The assay method was done according to the protocol ofSuigura, et al., 1979, Chem. Pharm. Bull. 27(9):2003-2007. The assay isa two step reaction in which hydrogen peroxide is generated by oxalateoxidase in the first step then detected quantitatively by a peroxidaselinked color reaction in the second. The color reaction is then measuredby spectrophotometer using visible light at 550 nm. The first step ofthe assay was initiated by grinding shoot derived leaf tissue, pooledleaf tips of 1 sample per shoot, in 0.1 M succinate buffer, pH 3.5. Theextracts were centrifuged and supernatants were discarded because mostof the enzyme activity is in the cell wall due to the signal peptide ofoxalate oxidase. The pellet was resuspended in 0.1 M succinate buffer,pH 3.5, and 0.05 ml of an oxalic acid solution consisting of 10 mMoxalic acid dissolved in 0.1 M succinate buffer, pH 3.5. The oxalateoxidase enzyme reaction proceeded with mild agitation at roomtemperature (25° C.) for 4 hr. At the end of this time period thereactions were centrifuged and an aliquot of the supernatant removed andadded to a volume of 1 M Tris, pH unadjusted, to adjust the samples to afinal pH of 7.0 (Tris to 0.147 M) for the second reaction step of theassay. Color development was done by adding the following components in0.2 M Tris HCl, pH 7.0, in a mixture such that listed finalconcentrations were achieved: horseradish peroxidase (20 μ/ml),4-aminoantipyrine (0.165 mM), and N,N-dimethylaniline (0.33 mM).Absorbance at 500 nm was read for samples of the color developmentreaction. Shoots positive for oxalate oxidase activity were moved intonodal culture for plant recovery and the negative shoots were discarded.

Positive shoots were divided into nodal explants where each explantcontained at least one node from which a shoot might be recovered. Nodalexplants were culture for 3 d on 374G medium (374E plus 250 mg/lcefotaxime) in the light to release nodal meristems then transferred to374C medium and cultured in the light at 26° C. for 4 weeks to allownodal shoot development. Shoots derived from nodal culture were assayedfor oxalate oxidase activity as described above. The oxalate oxidasepositive shoots were moved to procedures for plant recovery in thegreenhouse and the negatives were discarded.

Assay positive shoots were recovered by grafting to Pioneer sunflowerhybrid 6150 grown aseptically and in-vitro on 48 P medium (½×MS salts,0.5% sucrose, pH 5.0, 0.3% gelrite). Root-stock was prepared by surfacesterilizing seed of 6150 as described above for SMF3 then imbibing inthe light at 26° C. for 4 days. Following this initial germination step,seedlings are place in the dark on 48P medium for 4 d to elongatehypocotyls. The seedlings were then placed back into the light and couldbe used in the next 7-10 days for grafting. Grafting was done by firstcutting the 6150 seedling in the hypocotyl region below the meristemthen slicing the hypocotyl longitudinally in half at the cut site.Transgenic shoots are cut at their base to separate from the originatingexplant and secured on the root-stock by using a parafilm wrap. Afterabout one week in-vitro, the grafted plants were transferred to soil andmaintained under humid conditions until they could survive in drier airin the greenhouse.

Transformed T0 plants are further characterized by oxalate oxidaseactivity assays to verify the continued presence of an active transgeneand to determine if the transgene would be present in floral tissue. Ifthere is a sector of transformation which did not develop into a newportion of the growing T0 plant, that plant portion is trimmed off toinduce floral bud initiation from axillary meristems. T0 flowers areselfed, T1 seed is recovered, and the T1 seed is germinated for T1transgenic plant identification. Cotyledon or leaf tissue of T1seedlings is sampled and used to assay for the scorable transgene.

Example 5 Ectopic Expression of Soybean WUS to Induce Organogenesis

In addition to testing WUS in meristem transformation, other tissueexplants can be tested for the formation of adventive meristemsfollowing stable or transient transformation by WUS. The explant typesare well known in the art of dicot transformation and might includehypocotyl explants, leaf explants, cotyledon explants, or immaturetissues such as embryo or primary leaf as described here for sunflower.As described for meristem explants, the DNA delivery can be done byeither the direct delivery of particle bombardment or by Agrobacteriumdelivery by T-DNA. Using sunflower genotype SMF3 as an example, primaryleaves are isolated from meristem explants prepared as described above.After the overnight culture of dissected seeds on 374E medium, theprimary leaves have elongated. These are removed and placed in thecenter of sterile petri plates on filters moistened with 530 medium (MSsalts, B5 vitamins, 3% sucrose, 4 mg/l p-chlorophenoxyacetic acid, pH5.8) in preparation for particle bombardment. Primary leaf explants arespread out over the center of these plates such that none areoverlapping others. Particle bombardment is done exactly as describedabove for direct DNA delivery to meristem explants except that a sterile70 um nitex mesh is placed over the top of the explants to help preventthem from shifting during bombardment. The DNA delivered could include achimeric gene, consisting of a constitutive promoter such as SCP1combined with the selectable marker NPTII and the PINII 3′ region, thatallows for the preferential growth of transformed tissue. Alternatively,the WUS gene may provide a growth advantage to the tissue such that aselectable marker is not required. Following particle bombardment, theexplants are cultured for 3d on filters continuously moistened with 530medium by adding 0.5 mL of additional liquid medium per 24 hr. They arecultured in the Percival growth chamber in the light under 18 hrdaylength and at 26° C. Primary leaf explants that have shown growth arethen transferred to 374E medium containing 50 mg/l kanamycin if theselectable marker gene was used and cultured for 2 to 3 wk to allowtransgenic callus and shoot formation. Cultures that do not respond aretransferred every two weeks to 374E with 50 mg/l kanamycin untilrecoverable shoots are formed. Shoots are sampled, selected, andrecovered to the greenhouse as described for meristem explants above.

Sunflower primary leaves can be transformed using Agrobacterium byslight modifications to the protocols above. The explants on 530 mediumare bombarded as described for meristem explants in the Agrobacteriumprocedure above. An Agrobacterium suspension is produced exactly asdescribed for meristem explants except that the liquid culture is 25 mlinstead of 5 ml. The Agrobacterium cells are centrifuged, the growthmedium supernatant discarded, and the cells resuspended to a calculatedOD600 of 0.6 in inoculation medium. Primary leaf explants are inoculatedin this suspension for 10 min, then placed back on 530 medium andco-cultivated for 3 d under the growth chamber conditions describedabove. The explants are then transferred to 374D medium (374E, 50 mg/lkanamycin, 250 mg/l cefotaxime) and cultured for 2-3 weeks. Explants canbe transferred every two weeks to fresh 374D medium until shoots can berecovered.

Example 6 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding the instant polypeptide insense orientation with respect to the maize 27 kD zein promoter that islocated 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or SmaI) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes NcoI and SmaI and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366. The DNA segment from pML103contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zeingene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kDzein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA canbe ligated at 15° C. overnight, essentially as described (Maniatis). Theligated DNA may then be used to transform E. coli XL1-Blue (EpicurianColi XL-1 Blue™; Stratagene). Bacterial transformants can be screened byrestriction enzyme digestion of plasmid DNA and limited nucleotidesequence analysis using the dideoxy chain termination method (Sequenase™DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid constructwould comprise a chimeric gene encoding, in the 5′ to 3′ direction, themaize 27 kD zein promoter, a cDNA fragment encoding the instantpolypeptide, and the 10 kD zein 3′ region.

The chimeric gene described above can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept inthe dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton™ flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains bialophos (5 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containingbialophos. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing thebialophos-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 7 Transformation and Regeneration of Maize Embryos

Immature maize embryos from greenhouse donor plants are bombarded with aplasmid containing the gene of the invention operably linked to apromoter; this could be a weak promoter such as nos, an induciblepromoter such as In2, or a strong promoter such as Ubiquitin plus aplasmid containing the selectable marker gene PAT (Wohlleben et al.,1988, Gene 70:25-37) that confers resistance to the herbicide Bialaphos.Transformation is performed as follows.

Maize ears are harvested 8-14 days after pollination and surfacesterilized in 30% Chlorox bleach plus 0.5% Micro detergent for 20minutes, and rinsed two times with sterile water. The immature embryosare excised and placed embryo axis side down (scutellum side up), 25embryos per plate. These are cultured on 560 L medium 4 days prior tobombardment in the dark. Medium 560 L is an N6-based medium containingEriksson's vitamins, thiamine, sucrose, 2,4-D, and silver nitrate. Theday of bombardment, the embryos are transferred to 560 Y medium for 4hours and are arranged within the 2.5-cm target zone. Medium 560Y is ahigh osmoticum medium (560L with high sucrose concentration).

A plasmid vector comprising the gene of the invention operably linked tothe selected promoter is constructed. This plasmid DNA plus plasmid DNAcontaining a PAT selectable marker is precipitated onto 1.1 μm (averagediameter) tungsten pellets using a CaCl₂ precipitation procedure asfollows: 100 μl prepared tungsten particles in water, 10 μl (1 μg) DNAin TrisEDTA buffer (1 μg total), 100 μL 2.5 M CaCl₂, 10 μl 0.1 Mspermidine.

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 ml 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

The sample plates are positioned 2 levels below the stopping plate forbombardment in a DuPont Helium Particle Gun. All samples receive asingle shot at 650 PSI, with a total of ten aliquots taken from eachtube of prepared particles/DNA. As a control, embryos are bombarded withDNA containing the PAT selectable marker as described above without thegene of invention.

Following bombardment, the embryos are kept on 560Y medium, an N6 basedmedium, for 2 days, then transferred to 560R selection medium, an N6based medium containing 3 mg/liter Bialaphos, and subcultured every 2weeks. After approximately 10 weeks of selection, selection-resistantcallus clones are sampled for PCR and activity of the gene of interest.In treatments containing the WUS gene, growth is stimulated andtransformation frequencies increase, relative to the control. Positivelines are transferred to 288J medium, an MS based medium with lowersucrose and hormone levels, to initiate plant regeneration. Followingsomatic embryo maturation (2-4 weeks), well-developed somatic embryosare transferred to medium for germination and transferred to the lightedculture room. Approximately 7-10 days later, developing plantlets aretransferred to medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to Classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored for expression of the gene of interest.

Example 8 Ectopic Expression of Maize WUS to Induce Organogenesis

Using the genotype High type II as an example, embryos are isolated andcultured on 560P medium for 3-5 days. Twelve hours before bombardmentthese embryos are transferred to high osmotic 560Y medium. Expressioncassettes containing the WUS cDNA are then co-introduced into thescutella of these embryos along with an expression cassette containingthe Bar or Pat gene using methods well described in the art for particlegun transformations. Twelve to 24 hours following bombardment embryosare then transferred back to 560P culture medium and incubated in thedark at 26° C. After one week of culture these embryos are moved to 560Rselection medium. Cultures are then transferred every two weeks untiltransformed colonies appear. Expression of WUS will stimulate adventivemeristem (shoot) formation. This will be apparent when the cultures arecompared to controls (transformed without the WUS cDNA or non-induced).Using either inducible expression cassettes, tissue specific promoters,or promoters of varying strengths it will be possible to control thelevels of expression to maximize the formation of adventive meristems.Using either non-responsive genotypes or sub-optimal culture conditionswith responsive genotypes, only the transformed cells expressing the WUSc-DNA will form meristems and regenerate plants. For experiments inwhich WUS-induced shoot proliferation occurs via ectopic meristemformation, WUS can be used as a positive selective phenotype and noselection agent is required in the media. In this manner the WUS genecan be used as a positive selective marker (only the cells expressingthe c-DNA will form shoot meristems) and transformants can be recoveredwithout a negative selective agent (i.e. bialaphos, basta, kanamycin,etc.).

Example 9 Transient Expression of the WUS Gene Product to Induce ShootOrganogenesis

It may be desirable to “kick start” meristem formation by transientlyexpressing the WUS genes product. This can be done by delivering WUS 5′capped polyadenylated RNA, expression cassettes containing WUS DNA, orWUS protein. All of these molecules can be delivered using a biolisticsparticle gun. For example 5′ capped polyadenylated WUS RNA can easily bemade in vitro using Ambion's mMessage mMachine kit. Following theprocedure outlined above, RNA is co-delivered along with DNA containingan agronomically useful expression cassette. The cells receiving the RNAwill immediately form shoot meristems and a large portion of these willhave integrated the agronomic gene. Plants regenerated from theseembryos can then be screened for the presence of the agronomic gene.

Example 10 Maize Meristem Transformation

Meristem transformation protocols rely on the transformation of apicalinitials or cells that can become apical initials followingreorganization due to injury or selective pressure. The progenitors ofthese apical initials differentiate to form the tissues and organs ofthe mature plant (i.e., leaves, stems, ears, tassels, etc.). Themeristems of most angiosperms are layered with each layer having its ownset of initials. Normally in the shoot apex these layers rarely mix. Inmaize the outer layer of the apical meristem, the L1, differentiates toform the epidermis while descendents of cells in the inner layer, theL2, give rise to internal plant parts including the gametes. Theinitials in each of these layers are defined solely by position and canbe replaced by adjacent cells if they are killed or compromised.Meristem transformation frequently targets a subset of the population ofapical initials and the resulting plants are chimeric. If for example, 1of 4 initials in the L1 layer of the meristem are transformed only ¼ ofepidermis would be transformed. Selective pressure can be used toenlarge sectors but this selection must be non-lethal since large groupsof cells are required for meristem function and survival. Transformationof a meristem cell with a WUS sequence under the expression of apromoter active in the apical meristem (either meristem-specific orconstitutive) would allow the transformed cells to re-direct theinitiation of new apical initials driving the meristem towardshomogeneity and minimizing the chimeric nature of the plant body. Todemonstrate this, the WUS sequence is cloned into a cassette with apromoter that is active within the meristem (i.e. either a strongconstitutive maize promoter such as the ubiquitin promoter including thefirst ubiquitin intron, or a promoter active in meristematic cells suchas the maize histone, cdc2 or actin promoter). Coleoptilar stage embryosare isolated and plated meristem-up on a high sucrose maturation medium(see Lowe et al., 1997, In Genetic Biotechnology and Breeding of Maizeand Sorghum, AS Tsaftaris, ed., Royal Society of Chemistry, Cambridge,UK, pp 94 97). The WUS expression cassette along with a reporterconstruct such as Ubi:GUS:pinII can then be co-delivered (preferably 24hours after isolation) into the exposed apical dome using conventionalparticle gun transformation protocols. As a control, the WUS constructcan be replaced with an equivalent amount of pUC plasmid DNA. After aweek to 10 days of culture on maturation medium the embryos can betransferred to a low sucrose hormone-free germination medium. Leavesfrom developing plants can be sacrificed for GUS staining. Transientexpression of the WUS sequence in meristem cells, through formation ofnew apical initials, will result in broader sectors or completelytransformed meristems increasing the probability of germ-linetransformation. Integration and expression of the WUS sequence willimpart a competitive advantage to expressing cells resulting in aprogressive enlargement of the transgenic sector. Due to the WUS-inducedmaintenance of apical initials and growth of their transformedderivatives, they will supplant wild-type meristem cells as the plantcontinues to grow. The result will be both enlargement of transgenicsectors within a given cell layer (i.e. periclinal expansion) and intoadjacent cell layers (i.e. anticlinal invasions). As cells expressingthe WUS gene occupy an increasingly large proportion of the meristem,the frequency of transgene germline inheritance goes up accordingly.Using WUS in this manner to target meristems will increasetransformation rates, relative to control treatments. Coleoptilar-stageembryos used as a source of meristems is used as an example, but othermeristem sources could be used as well, for example immatureinfluorescences.

Example 11 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), Sma I, Kpn I and Xba I.The entire cassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embryos may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can be maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with fluorescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptide and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 12 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% low melting agarose gel. Buffer and agarose contain 10μg/ml ethidium bromide for visualization of the DNA fragment. Thefragment can then be purified from the agarose gel by digestion withGELase™ (Epicentre Technologies, Madison, Wis.) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptide are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21 (DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

Example 13 Use of Flp/Frt System to Excise the WUS Cassette

In cases where the WUS gene has been integrated and WUS expression isuseful in the recovery of maize trangenics (i.e. under conditions wherecontinuous expression of WUS promotes adventive meristem formation), butis ultimately not desired in the final product, the WUS expressioncassette (or any portion thereof that is flanked by appropriate FRTrecombination sequences) can be excised using FLP-mediated recombination(see U.S. patent application Ser. No. 08/972,258 filed Nov. 18, 1997).

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference.

1. An isolated polynucleotide comprising: a nucleotide sequence encodingthe WUSCHEL polypeptide of SEQ ID NO: 6 or SEQ ID NO: 8 which stimulatesin vitro growth of plant tissue.
 2. The isolated polynucleotide of claim1, wherein the nucleotide sequence comprises SEQ ID NO:5, or SEQ IDNO:7.
 3. An isolated polynucleotide comprising the complement of thepolynucleotide of any one of claims 1 and 2, wherein the complement andthe polynucleotide consist of the same number of nucleotides and are100% complementary.
 4. A chimeric gene comprising the polynucleotide ofany one of claims 1 and 2, operably linked to a regulatory sequence. 5.A transgenic plant comprising the polynucleotide of claim
 1. 6. Thetransgenic plant of claim 5, wherein the plant is corn, soybean, wheat,rice, alfalfa, sunflower, canola, or cotton.
 7. A seed from thetransgenic plant of claim 5, wherein the seed comprises thepolynucleotide.
 8. The seed of claim 7, wherein the seed is from corn,soybean, wheat, rice, alfalfa, sunflower, canola, or cotton.
 9. A methodfor inducing meristem proliferation in a plant cell comprising: (a)transforming a plant cell with the polynucleotide of claim 1 operablylinked to a regulatory sequence operable in the plant cell; and, (b)expressing the polynucleotide to induce meristem proliferation.
 10. Themethod of claim 9 wherein the polynucleotide is integrated into theplant cell genome to produce a transformed plant cell comprising thepolynucleotide.
 11. The method of claim 10 further comprising growingthe transformed plant cell under plant growing conditions to produce aregenerated plant.
 12. A plant produced by the method of claim 11,wherein the plant comprises the polynucleotide.
 13. The plant of claim12, wherein the plant is corn, soybean, wheat, rice, alfalfa, sunflower,canola, or cotton.
 14. A method for positive selection of a transformedcell, comprising: (a) transforming a plant cell with the polynucleotideof claim 1 operably linked to a regulatory sequence operable in theplant cell, and (b) expressing the polynucleotide for a time sufficientto induce organogenesis and provide a positive selection means.
 15. Amethod for transforming a plant cell comprising introducing thepolynucleotide of any one of claims 1 and 2 into the cell.
 16. Thetransformed plant cell produced by the method of claim 15, wherein theplant cell comprises the polynucleotide.
 17. A method for transforming aplant cell comprising introducing the polynucleotide of claim 3 into thecell.
 18. The transformed plant cell produced by the method of claim 17,wherein the plant cell comprises the polynucleotide.
 19. The method ofclaim 15, further comprising growing the transformed plant cell underplant growing conditions to produce a regenerated plant.
 20. The methodof claim 17, further comprising growing the transformed plant cell underplant growing conditions to produce a regenerated plant.
 21. Atransformed plant produced by the method of claim 9, wherein the plantcomprises the polynucleotide.
 22. A transformed plant produced by themethod of claim 20, wherein the plant comprises the polynucleotide. 23.The plant of claim 21, wherein the plant is corn, soybean, wheat, rice,alfalfa, sunflower, canola, or cotton.
 24. The plant of claim 22,wherein the plant is corn, soybean, wheat, rice, alfalfa, sunflower,canola, or cotton.